HIGHER-AFFINITY OLIGOSACCHARIDE LIGANDS FOR E-SELECTIN

A series of synthetic oligosaccharides based on sialyl Lewis x (sLe(x); Neu5Acalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc) and sialyl Lewis a (sLe(a); Neu5Acalpha2-3Galbeta1-3[Fucalpha1-4 ]GlcNAc) was used to study the binding interactions of selectins. E-selectin-immunoglobulin fusion protein (E-selectin-Ig) bound to immobilized bovine serum albumin (BSA)-neoglycoproteins containing sLe(x) or sLe(a) in a Ca2+-dependent manner. Solution-phase sLe(x) tetrasaccharide blocked this interaction by 50% at a concentration of 750+/-20 muM (IC50). sLe(a) was more effective (IC50 = 220+/-20 muM), while nonsialylated, nonfucosylated derivatives showed little or no activity at concentrations up to 1 mM. Attachment of an 8-methoxycarbonyloctyl aglycone in a beta linkage to the anomeric carbon of the GlcNAc of sLe(x) or sLe(a) increased their blocking activity nearly twofold. Finally, replacement of the 2-N-acetyl substituent of the GlcNAc by an azido or amino group resulted in substantial increases in activity, with the most potent inhibitor being amino substituted sLe(a), which was 36-fold more active (IC50 = 21+/-3 muM) than the reducing tetrasaccharide sLe(x). In contrast to results obtained with E-selectin-Ig, P-selectin-Ig binding to immobilized BSA-sLe(a) was blocked modestly by most oligosaccharides at 1 mM, with no substantial differences among them. IC50 values of soluble oligosaccharides determined in competitive binding studies accurately predicted blocking of leukocyte adhesion to recombinant E-selectin-Ig and to cytokine-activated endothelium.